Hi,
I have one expt problem. tht needs some troubleshooting.
I want to express some cytosolic proteins (1000 amino acids) on cell
surface. so i can use the cells for my expts.
Construct design :
N-terminus flag tag, a signal peptide ( from ICAM molecule) followed by a
cloning site and a trans-membrane domain with a short tail (from ICAM) and
2A- GFP.
there are lot of glycines in between to help for proper folding. i use 10+
amino acids more on either side of predicted trans-membrane domain so as to
help for proper folding.
problems:
1.Without the signal peptide but with trans-membrane domain, i see flag tag
on surface of GFP +ve cells but with signal peptide no flag tag staining.
which is understandable as signal is chopped off along with flag tag. hence
no staining.
2. Construct with out signal peptide but with membrane and gene of
interest, i donot see the expression of protein or FLAG in gfp +ve cells.
hence FLAG tag is not present or not put into surface.
The probable reasons:
-> proteins are toxic hence degraded.
-> signal peptide not strong to guide protein to membrane. any other strong
signal peptide or change in position of signal peptide in construct.
If any one has any experience/ suggestions are welcome
--
With regds
Itisam